The studies in this application seek to elucidate the control of neuroendocrine differentiation in normal calcitonin producing thyroid C-cells and their neoplastic counterpart, medullary thyroid carcinoma (MTC). Understanding this control could provide important insight for events in tumor development and progression. Medullary thyroid carcinoma is a good model for this study, since defined stages of progression which involve loss of neuroendocrine differentiation features, including capacity for calcitonin production, have been identified. We have developed an in vitro system to study steps of MTC differentiation and progression. MTC cells in culture can be differentiated with respect to many characteristics of normal C-cells, including increased calcitonin gene expression, either chemically or by ras or raf oncogene introduction. The current proposal will further characterize these findings, by cell biology, biochemistry, and molecular biology, concentrating on the molecular mechanisms regulating the induced differentiation. The studies proposed will be important not only for identifying the processes which may be involved in MTC progression, but also for defining the factors and pathways involved in neuroendocrine differentiation, and further elucidating the mechanisms of ras and raf oncogene function in cells. 1. Two cloned proteins, which bind specifically to the ras/raf responsive elements of the calcitonin gene, will be characterized for their function as transcription factors. The mechanisms of regulation which may allow these proteins, in MTC and other cell types, to mediate phenotypic responses to ras or raf (differentiation or transformation) will be examined. 2. The ret oncogene, which is activated by mutation in hereditary MTC, and in many cases of sporadic MTC, is silenced during raf-mediated differentiation of MTC cells in culture. The mechanisms of this silencing will be explored. The possibility will be tested that ret silencing is a necessary or sufficient step for MTC terminal differentiation. This could have important therapeutic implications in MTC. It will be determined if ret can be silenced in other cell types, and if other tyrosine kinases can be silenced by similar mechanisms. If other tyrosine kinases can be silenced, this could also provide a therapeutic target in several common cancers, in which overexpression of tyrosine kinases contributes to cancer growth.